Chemical probes for left-handed DNA and chiral metal complexes as Z-specific anti-tumor agents

ABSTRACT

This invention concerns a coordination complex and salts and optically resolved enantiomers thereof, of the formula (R) 3  --M, wherein R comprises 1,10-phenanthroline or a substituted derivative thereof, M comprises a suitable transition metal, e.g. ruthenium(II) or cobalt(III), and R is bonded to M by a coordination bond. 
     The complexes of this invention are useful in methods for labeling, nicking and cleaving DNA. The lambda enantiomer of complexes of this invention is useful in methods for specifically labeling, detecting, nicking and cleaving Z-DNA. 
     The complexes may also be used in a method for killing tumor cells and may be combined with a pharmaceutically acceptable carrier to form a pharmaceutical composition for the treatment of tumor cells in a subject. The invention further concerns methods for treating a subject afflicted with tumor cells.

This invention was made with government support under grant numbers GM32203 and GM 33309 from the National Institutes of Health, United StatesDepartment of Health and Human Services, and grant number CHE-83-51017from the National Science Foundation. The government has certain rightsin this invention.

This is a division of application Ser. No. 693,023, filed Jan. 18, 1985now U.S. Pat. No. 4,721,669.

BACKGROUND OF THE INVENTION

Much of the information set forth herein has been published. See Barton,J. K. et al., J. Am. Chem. Soc., 1984, 106: 2172-2176 (Apr. 6, 1984);Barton, J. K. and Raphael, A., J. Am. Chem. Soc., 1984, 106: 2466-2468(Apr. 18, 1984); Barton, J. K. et al., Proc. Natl. Acad. Sci. USA, 1984,81:1961-1965 (Apr. 27, 1984); and Barton, J. K., J. Biom. Structure andDynam., 1983, 1: 621-632 (Jan. 18, 1984). The above-mentioned paperswere distributed by the respective publishers on the dates provided inparentheses.

Throughout this application various publications are referenced byarabic numerals within parentheses. Full citations for thesepublications may be found at the end of the specification immediatelypreceding the claims. The disclosures of these publications in theirentireties are hereby incorporated by reference into this application inorder to more fully describe the state of the art to which thisinvention pertains.

The binding of heterocyclic compounds to DNA by intercalation, where theplanar aromatic cation stacks between adjacent base pairs of the duplex(1), has been the subject of considerable investigation (2-4).

Intercalative drugs can be strongly mutagenic, and some, as adriamycinand daunomycin, serve as potent chemotherapeutic agents (5). The smallintercalators such as ethidium or proflavine in addition provide usefulchemical probes of nucleic acid structure (6). Metallointercalators havebeen particularly useful in probing DNA structure and the intercalationprocess itself, because the ligands or metal may be varied in an easilycontrolled manner to facilitate the individual application (7).

The aromatic chromophore of the intercalative cation can provide asensitive handle to monitor the conformation and flexibility of thehelix. Many intercalators show antibacterial or anticancer activity and,because the inserted residue often resembles a base pair in shape andthickness, intercalators are commonly frame shift mutagens. (3)Intercalation appears to require, simply, a planar heterocyclic residue,(4) and in fact cationic metal complexes which contain aromatic ligandsbind to DNA by intercalation as well. (5) Platinum intercalators haveuniquely provided electron dense probes for x-ray diffractionexperiments. (6) Moreover the metallointercalation reagents have offeredparticular experimental flexibility in that both the metal and accessoryligands may be varied for individual applications. Comparisons of thebinding of the intercalative 2,2'-bipyridylplatinum(II) reagent with theanalogous nonintercalating bis(pyridine)platinum(II) species by fiberX-ray diffraction methods, for example, demonstrated quite simply therequirement for ligand planarity in the intercalation process (8).

The original studies of metallointercalators centered on square-planarplatinum(II) complexes containing aromatic terpyridyl or phenanthrolineligands (9), and single-crystal studies of terpyridylplatinum(II)species stacked with nucleotides showed the platinum complex to insertalmost fully between the base pairs (10,11). More recently the reagentmethidiumpropyl-Fe(II) EDTA, which contains a redox-active metal centertethered to an organic intercalator, has been applied in "footprinting"experiments to determine the sequence specificities of small drugs boundto DNA (12). Bis(phenanthroline)-cuprous ion (13) has similarly beenemployed in DNA cleavage experiments (14), and this reagent alsopresumably birds initially to the DNA by intercalation.(3,5,6,8-tetramethyl-1,10-phenanthroline)₃ Ru(II) has been reported andits use against influenza virus, fungus, yeast and leukemia investigated(98). The ability of the tris tetramethyl complex to bind to or cleaveDNA has not been reported. Furthermore, enantiomers of that complex havenot been separated and their respective properties compared.

Reagents of high specificity and even stereoselectivity would bedesirable in the design both of potent drugs and of structural probes.For the chiral complex (phen)₃ Zn²⁺ (phen=1,10-phenanthroline) anenantiomeric preference in binding to B-DNA has been observed (15). Asfor the tetrahedral (phen)₂ Cu⁺ complex, and in contrast to thesquare-planar platinum intercalators, the octahedral coordination in thetris(phenanthroline) metal cations can permit a partial insertion ofonly one coordinated ligand. Thus while one ligand is stacked betweenbase pairs, the remaining nonintercalated phenanthroline ligands shouldbe available to direct the enantiomeric selection.

The left-handed DNA helix has received considerable attention since theoriginal crystallographic study of the Z-DNA fragment [d(CpG)]₆ (16).Solution conditions that include high ionic strength (17), hydrophobicsolvents (18), the presence of certain trivalent cations (19), orcovalent modification with bulky alkylating agents (19-23) allfacilitate the transition of a right-handed double helix into aleft-handed form. This striking conformational transition was firstobserved for poly[d(G-C)] (17). More recently, the alternatingpurine-pyrimidine sequence [d(G-T)]_(n).[d(C-A]_(n) has been shown toform Z-helices as well (24, 25). Methylation of cytosine residues atcarbon-5 lends stability to Z-form DNA (19, 26) and, under physiologicalconditions, transitions to a left-handed structure can occur to relievethe torsional strain in underwound negatively supercoiled DNA (27-29).These latter findings suggest mechanisms for left-handed DNA formationin the cell, where such structures could be important in controllinggene expression. Negatively supercoiled simian virus 40 DNA, forexample, has been found to contain potentially Z-DNA-forming alternatepurine-pyrimidine regions within transcriptional enhancer sequences(30).

To explore any biological role for left-handed DNA, sensitive andselective probes are required. Assays of superhelix unwinding, NMRexperiments and circular dichroism have been used in detecting Z-DNA.These methods, however, are indirect, do not assay for helix handedness,and require large quantitites of material. More recently antibodies toZ-DNA have been elicited. The antibodies provide a more sensitive meansof detection. Z-DNA appears to be a strong immunogen. Anti-Z-DNAantibodies have been elicited with both brominated poly[d(G-C)] (31) andpoly[d(G-C)] modified with diethylenetriamineplatinum(II) (32) asantigens. The structures of Z-DNA and in particular of a modified Z-formprovide a multitude of antigenic characteristics: the left-handedhelicity, the zigzag dinucleotide phosphate repeat, the protrudingpurine substituents in the shallow major groove. It is not surprisingthen that the various antibodies obtained appear specific for differentlocalized features of Z-DNA (33). The development of a specific chemicalprobe, so designed as to recognize a known structural element of Z-DNA,e.g. the helix handedness, would offer a simple complementary approachbut has not heretofore been reported.

Enantiomeric selectivity has been observed in the interactions oftris(phenanthroline) metal complexes with B-DNA (15, 34-35). Experimentswith tris(phenanthroline)zinc(II) have indicated stereoselectivity (15);dialysis of B-DNA against the racemic mixture leads to the opticalenrichment in the Λ enantiomer. Subsequent luminescence,electrophoretic, and equilibrium dialysis studies of thewell-characterized ruthenium(II) analogues have shown that thetris(phenanthroline) metal isomers bind to DNA by intercalation and itis the Δ enantiomer that binds preferentially to a right-handed duplex(34,35). The enantiomeric selectivity is based on steric interactionsbetween the nonintercalated phenanthroline ligands and the phosphatebackbone. Although the right-handed propeller-like isomer intercalateswith facility into a right-handed helix, steric repulsions interferewith a similar intercalation of the Λ enantiomer.

Based on this premise, tris(phenanthroline) metal complexes appearuseful in the design of probes to distinguish left-handed andright-handed DNA duplexes. The design flexibility inherent inmetallointercalation reagents, in which both ligand and metal may bevaried easily, makes the coordination complexes attractive probes(7,8,35). We have concentrated here on phenanthroline complexes ofruthenium(II) because of the high luminescence associated with theirintense metal-to-ligand charge-transfer band (37, 38) and because theexchange-inert character of the low-spin d⁶ complexes limitsracemization (20).

Furthermore, there has been considerable interest in DNA endonucleolyticcleavage reactions that are activated by metal ions, (39,40) both forthe preparation of "footprinting" reagents (41) and as models for thereactivity of some antitumor antibiotics, notably bleomycin (42) andstreptonigrin. (43) The features common to these complexes are that themolecule has a high affinity for double-stranded DNA and that themolecule binds a redox-active metal ion cofactor. The delivery of highconcentrations of metal ion to the helix, in locally generating oxygenor hydroxide radicals, yields an efficient DNA cleavage reaction.Additionally, cobalt(III) bleomycin cleaves DNA in the presence oflight.(44)

SUMMARY OF THE INVENTION

This invention concerns a coordination complex or salt thereof havingthe formula (R)₃ ---Co(III), wherein R comprises 1,10-phenanthroline ora substituted derivative thereof and R is bound to Co by a coordinationbond.

One embodiment concerns a method for labeling DNA with a complex of theformula (R)₃ ---M, wherein R comprises 1,10-phenanthroline or asubstituted derivative thereof, M comprises a suitable transition metal,and R is bonded to M by a coordination bond. In this and otherembodiments a suitable transition metal is one which is capable offorming an octahedral complex with R, such as ruthenium (II) or cobalt(III). The invention also concerns a DNA molecule labeled with a complexof the formula (R)₃ ---M, as defined above, wherein the complex is boundto the DNA by intercalation.

Another embodiment of this invention is a method for selectivelylabeling Z-DNA with the lambda enantiomer of a complex of the formula(R)₃ ---M, as defined above. This method comprises contacting the lambdaenantiomer of the complex under suitable conditions such that thecomplex binds to the Z-DNA. The invention further involves a labeled DNAmolecule comprising Z-DNA and the lambda enantiomer of a complex of theformula (R)₃ ---M, as defined above, the complex being bound to theZ-DNA.

Another embodiment of this invention concerns a method for detecting thepresence of Z-DNA. This method involves selectively labeling Z-DNAaccording to the above-mentioned method and detecting the presence ofthe complex bound to the Z-DNA.

Still another embodiment of this invention is a method for nickingdouble-stranded DNA by effecting breakage of at least one phosphodiesterbond along the DNA. The method involves contacting the DNA with a cobalt(III)-containing complex of this invention under suitable conditionssuch that the complex intercalates into the DNA to form an adduct andirradiating the adduct so formed with a sufficient dose of ultravioletradiation of an appropriate wavelength so as to nick the DNA at thesite(s) of intercalation. This invention further involves a method forcleaving double-stranded DNA which comprises nicking the DNA by theabove-mentioned method and treating the nicked DNA so produced with asuitable enzyme capable of cleaving single-stranded DNA under effectiveconditions to cleave the nicked DNA at the site of the nick(s).

An additional embodiment of this invention is a method for selectivelynicking Z-DNA by effecting breakage of at least one phosphodiester bondalong the Z-DNA. The method involves contacting a DNA moleculecontaining a Z-DNA sequence with a lambda enantiomer of a cobalt(III)-containing complex of this invention under suitable conditionssuch that the complex binds to the Z-DNA to form an adduct andirradiating the adduct so formed with a sufficient dose of ultravioletradiation of an appropriate wavelength so as to nick the DNA at thebinding site(s). Double-stranded Z-DNA may be selectively cleaved byselectively nicking the Z-DNA by the above-mentioned method and treatingthe nicked DNA so produced with a suitable enzyme capable of cleavingsingle-stranded DNA under effective conditions to cleave the nickeddouble-stranded DNA at the site of the nick(s).

This invention further concerns a method for killing a portion of apopulation of appropriate tumor cells. The method involves contactingthe tumor cells under suitable conditions with an effective amount ofthe lambda enantiomer of a coordination complex of the formula (R)³---M, as previously defined, to kill the tumor cells. In anotherembodiment a racemic cobalt(III)-containing complex of the invention maysimilarly be used to kill tumor cells. When a cobalt (III)-containingcomplex is used, the method may further comprise irradiating the tumorcells with a suitable dose of ultraviolet radiation of an appropriatewavelength at a suitable time after the tumor cells have been contactedwith the complex, permitting the complex to nick DNA.

Still another embodiment cf this invention is a pharmaceuticalcomposition for the treatment of tumor cells in a subject. Thepharmaceutical composition comprises a pharmaceutically acceptablecarrier and an effective anti-tumor amount of a cobalt(III)-containingcomplex of the invention or of the lambda enantiomer of a complex of theformula (R)₃ ---M, as defined previously. Such a composition may be usedin a method for treating a subject afflicted with tumor cells so as tocause regression of the tumor cells. This method involves administeringto the subject by a suitable route the pharmaceutical composition in anamount sufficient to cause regression of the tumor cells.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Micrographs of Drosophila polytene chromosomes stained withracemic RuDIP by phase contrast (left) and fluorescent (right)microscopy. High concentrations of RuDIP, bright regions in thefluorescent micrographs, are found at the band regions (the dark regionsby phase contrast).

FIG. 2. (a) Visible absorption spectra of racemic (phen)₃ Ru²⁺ (50 μM)in the absence (--) and presence (---) of DNA (1 mM). (b) Luminescencespectra of free (---) (phen)₃ Ru²⁺ and of Λ-(phen)₃ Ru²⁺ (.sup.. . .)and Δ-(phen)₃ Ru²⁺ (--) in the presence of DNA (0.3 mM). Rutheniumconcentrations were 10 μM. Sample excitation was at 447 nm.

FIG. 3. Relative mobilities of closed ( ) and nicked ( ○ ) circularpColEl DNA in the presence of increasing concentrations of added Δ-(above and Λ- (below) (phen)₃ Ru²⁺. Bars indicate the width of the DNAbands on the basis of the distribution of topoisomers. Comigrationpoints of nicked and closed circular DNAs are seen at 90 and 120 μM forthe Δ and Λ isomers, respectively.

FIG. 4. Scatchard plot of (phen)₃ Ru²⁺ binding to calf thymus DNA inbuffer 1 at 22° C., where r is the ratio of bound ruthenium tonucleotide concentrations and C is the concentration of ruthenium freein solution. The solid curve represents the best fit to eq 1.

FIG. 5. Circular dichroism of (□) Δ-(phen)₃ Ru²⁺ and of solutions afterdialysis of rac-(phen)₃ Ru²⁺ against (+) B-DNA and ( ) Z-DNA. Dialysisagainst B-DNA leads to enrichment of the solution in the unbound Λisomer.

FIG. 6. Visible absorption spectra of racemic RuDIP (5.3 μM) in theabsence and presence of various concentrations of B-form calf thymus DNA(Left) and Z-form poly[d(G-C)] (Right) in buffer 1.

FIG. 7. Luminescence spectra in buffer 2. . . . , Racemic RuDIP (3 μM)free in solution; ---, racemic RuDIP (3 μM) in the presence of Z-formpoly[d(G-C)]; --, B-form calf thymus DNA. Samples were excited at 482nm.

FIG. 8. Relative change in absorption intensity at 460 nm of racemicRuDIP (x), Λ-RuDIP ( ○ ), and Δ-RuDIP ( ○ ) at variousDNA-phosphate/ruthenium ratios. Titrations were conducted in buffer 1using either B-form calf thymus DNA (Upper) or Z-form poly[d(G-C)](Lower). Strong stereoselectivity with B-DNA is evident based on thedifferential hypochromism seen between enantiomers, whereas comparabletitrations with Z-DNA show the same hypochromic effects with eachenantiomer. Because large hypochromicity in the Λ isomer is seen onlywith Z-DNA, Λ-RuDIP provides a probe for this conformation.

FIG. 9. (A) Cleavage of plasmid ColEl DNA in the presence of (phen)₃Co³⁺ and light. The 1% agarose gel shows the distribution of DNA forms(100 μM nucleotide) initially without cobalt (lane 1) and afterirradiation at 254 nm (4-W mercury lamp) in the presence of 10 μMCo(phen)₃ ³⁺ for 0, 20, 40, and 60 min (lanes 2-5, left to right). Thesamples were incubated in 50 mM Tris acetate buffer, pH 7.0, and 18 mMNaCl and then electrophoresed for 1 h at 60 V and stained with ethidium.(B) The cleavage is also stereoselective. Plasmid ColEl DNA (100 μM) wasincubated with Λ- (left) or Δ-Co(DIP)₃ ³⁺ (right) (5 μM) and irradiatedfor 0, 0.5, 1.0, or 1.5 h (lanes 1-4 and 5-8 for the Λ and Δ isomers,respectively). Incubation of this DNA with Λ-Co(DIP)₃ ³⁺ in light has noeffect, while incubation with Δ-Co(DIP)₃ ³⁺ in light causes completeconversion of form I to form II.

FIG. 10. Plot showing the percent reduction in form I band intensity forpBR322 DNA (closed symbols) and pColEl DNA (open symbols) incubated witheither Δ-(triangles) or Λ-Co(DIP)₃ ³⁺ (circles) (5 μM) as a function oftime irradiated. Extensive strand scission is evident in pBR322 withboth isomers.

FIG. 11. Experimental scheme used to map the single-stranded cleavagesites of Co(DIP)₃ ³⁺.

FIG. 12. Densitometric scans of ∂-Co(DIP) fragmentation using the schemedepicted in FIG. 1. The double stranded fragments produced in pBR322using AvaI for linearization are the following sizes in kilobase pairs(kb): 3.5 and 0.75, 2.9 and 1.5, 2.6 and 1.8. Note that these pairs ofbands sum to 4.4 kb, the length of the plasmid. In pLP32, a pair ofbands not present in pBR322 are seen at 3.3 and 1.1 kb. These bandsindicate cleavage at the d(CG)₃₂ insert at position 375 (1.1 or 3.3 kbfrom the AvaI site at 1424). This plasmid was a gift of Dr. A. Nordheim.

Densitometric scans were performed on photographs using a Cary 219spectrophotometer with gel scanning attachment. Band sizes werequantitated using markers of known molecular weight. Note that since thebands were stained with ethidium bromide, the band intensities areweighted by their molecular weight.

FIG. 13. Coarse map of left-handed sites in pBR322. (a) Densitometricscans of Λ-Co(DIP)₃ ³⁺ fragmentation after linearization with EcoRI.Bands (in kilobase) are produced at 3.3 and 1.1, 2.75 and 1.45, and 2.2.Since EcoRI cleaves at position zero, these fragments correspond torecognition sites at 3.3, 1.45, and 2.2. (b) Biological map of pBR322showing positions of cleavage by Λ-Co(DIP)₃ ³⁺ (Λ).

FIG. 14. Schematic view of the enantiomers of (phen)₃ Ru²⁺ bound toB-DNA, illustrating the basis for stereo selectivity. Unfavorable stericinteractions are seen between the nonintercalated ligands of the Δisomer and the DNA phosphate backbone. In contrast the Δ isomer fitseasily in a right-handed helical groove.

FIG. 15. Corey-Pauling-Kolton space-filling models of Δ- and Λ-RuDIPwith a right-handed B-DNA helix. The orientation of the metal complex inthe helix is shown in the sketches. One ligand (not visible) is orientedperpendicular to the helix axis for intercalation between the basepairs. (A) Intercalation of the Δ enantiomer. The ronintercalatedligands fit easily within the right-handed groove. (B) For the Λenantiomer, when one ligand is positioned for intercalation, theremaining two ligands are blocked completely above and below (arrows) bythe right-handed sugar-phosphate backbone, and this steric constraintprevents ligand insertion between the base pairs. This can also be seenby following the line of the DNA backbone, which, although completelyvisible in A, is interrupted by the phenyl groups of Λ-RuDIP in B.

DETAILED DESCRIPTION OF THE INVENTION

As mentioned above, this invention concerns a coordination complex orsalt thereof having the formula (R)3---Co(III), wherein R comprises1,10-phenanthroline or a substituted derivative thereof and R is boundto Co by a coordination bond. A "substituted derivative" as the phraseis used herein is a compound obtained by replacing one or more hydrogenatoms present in 1,10-phenanthroline with one or more moieties havingthe characteristic that the complex containing the resulting compoundbinds to DNA. Merely by way of example, the substituted derivative of1,10-phenanthroline may be 4,7-diamino-1,10-phenanthroline;3,8-diamino-1,10-phenanthroline;4,7-diethylenediamine-1,10-phenanthroline;3,8-diethylenediamine-1,10-phenanthroline;4,7-dihydroxyl-1,10-phenanthroline;3,8-dihydroxyl-1,10-phenanththroline; 4,7-dinitro-1,10-phenanthroline;3,8-dinitro-1,10-phenanthroline; 4,7-diphenyl-1,10-phenanthroline;3,8-diphenyl-1,10-phenanthroline; 4,7-dispermine-1,10-phenanthroline, or3,8-dispermine-1,10-phenanthroline. Unless otherwise specified thecomplex of this invention is a racemic mixture of enantiomers. Theinvention also concerns the optically resolved delta and lamda isomersof the complex.

One embodiment concerns a method for labeling DNA with a complex of theformula (R)₃ ---M, wherein R comprises 1,10-phenanthroline or asubstituted derivative thereof as defined above, M comprises a suitabletransition metal, and R is bonded to M by a coordination bond. In thisand other embodiments a suitable transition metal is one which iscapable of forming an octahedral complex with R, such as ruthenium (II)or cobalt (III). The labeling method involves contacting the DNA withthe complex under suitable conditions such that the complex binds to theDNA, e.g. by intercalation.

The invention also concerns a DNA molecule labeled with a complex of theformula (R)₃ ---M, as defined above, and the complex is bound to theDNA, e.g. by intercalation. Preferably the labeled DNA molecule isproduced by the above-described method.

A further embodiment is a method for selectively labeling Z-DNA with thelambda enantiomer of a complex of the formula (R)₃ ---M, as definedabove. This method comprises contacting the DNA with the lambdaenantiomer of the complex under suitable conditions such that thecomplex binds to the Z-DNA. The invention further involves a labeled DNAmolecule comprising Z-DNA and the lambda enantiomer of a complex of theformula (R)₃ ---M, as defined above, the complex being bound to theZ-DNA. Preferably the labeled DNA molecule is produced by theabove-described method.

Another embodiment of this invention concerns a method for detecting thepresence of Z-DNA. This method involves selectively labeling Z-DNAaccording to the above-mentioned method and detecting the presence ofthe complex bound to the Z-DNA, e.g. by spectroscopic methods (SeeExperiments hereinafter).

Still another embodiment of this invention is a method for nickingdouble-stranded DNA by effecting single-stranded scission, i.e.,breakage of at least one phosphodiester bond along the DNA. The methodinvolves contacting the DNA with a cobalt (III)-containing complex ofthis invention under suitable conditions such that the complex binds tothe DNA, e.g. by intercalation, to form an adduct and irradiating theadduct so formed with a sufficient dose of ultraviolet radiation of anappropriate wavelength so as to nick the DNA at the site(s) ofintercalaticn. An appropriate ultraviolet wavelength in this an otherembodiments is a wavelength which is absorbed by the ligand bands of thecomplex used. As described hereinafter, the ligand band absorption of acomplex of this invention may be determined spectroscopically byconventional methods.

This invention further involves a method for cleaving double-strandedDNA which comprises nicking the DNA by the above-mentioned method andtreating the nicked DNA so produced with a suitable enzyme capable ofcleaving single-stranded DNA under effective conditions to cleave thenicked double-stranded DNA at the site of the nick(s). By this methoddouble-stranded scission of the DNA is effected. Suitable enzymes foreffecting double-stranded cleavage of nicked DNA in this and otherembodiments include those which are not deactivated in the presence ofthe complex used for DNA nicking, e.g. S1 nuclease.

An additional embodiment of this invention is a method for selectivelynicking Z-DNA by effecting breakage of at least one phosphodiester bondalong the Z-DNA. The method involves contacting a DNA moleculecontaining a Z-DNA sequence with the lambda enantiomer of a cobalt(III)-containing complex of this invention under suitable conditionssuch that the complex binds to the Z-DNA to form an adduct andirradiating the adduct so formed with a sufficient dose of ultravioletradiation of an appropriate wavelength, as previously defined, so as tonick the DNA at the binding site(s). Double-stranded Z-DNA may beselectively cleaved by selectively nicking the Z-DNA by theabove-mentioned method and treating the nicked DNA so produced with asuitable enzyme capable of cleaving single-stranded DNA under effectiveconditions to cleave the nicked DNA at the site of the nick(s).

This invention further concerns a method for killing a portion of apopulation of appropriate tumor cells. The method involves contactingthe tumor cells under suitable conditions with an effective amount ofthe lambda enantiomer of a coordination complex of the formula (R)³ --M,as previously defined, to kill the tumor cells. Alternatively, a racemiccobalt(III)-containing complex of this invention may be similarly used.When a cobalt (III)-containing complex is used, the method may furthercomprise irradiating the tumor cells with a suitable dose of ultravioletradiation of an appropriate wavelength at a suitable time after thetumor cells have been contacted with the complex, permitting the complexto nick DNA.

Still another embodiment of this invention is a pharmaceuticalcomposition for the treatment of tumor cells in a subject e.g. a humanor animal. The pharmaceutical composition comprises an effectiveanti-tumor amount of the lambda enantiomer of a complex of the formula(R)₃ ---M, as defined previously, and a pharmaceutically acceptablecarrier. Again, a racemic cobalt(III)-containing complex may be usedalternatively. Preferably the complex is a cobalt(III)-containingcomplex. Suitable carriers include steriIe saline or buffer-containingsolutions or other carriers known in the art such as those used withcisplatin.

Such a composition may be used in a method for treating a subject, e.g.a human or animal, afflicted with tumor cells so as to cause regressionof the tumor cells. This method involves administering to the subject bya suitable route the pharmaceutical composition in an amount sufficientto cause regression of the tumor cells. Suitable routes ofadministraiton include parenteral administration and topicaladministration, e.g. in cases such as skin cancers where the tumor cellsare located on or near an exposed surface of the subject. Furthermore,if the complex used is a cobalt(III)-containing complex, the method mayadditionally involve irradiating the tumor cells with a suitable dose ofultraviolet radiation of an appropriate wavelength permitting thecomplex to nick DNA. In this method the irradiation should be conductedat a suitable time after administration of the composition to thesubject, i.e. to permit the complex to interact with the DNA. It shouldalso be noted that optically resolved entantiomers of the complexes ofthis invention, specifically the lamboda isomer, may provide superiorresults both in killing tumor cells and in treating subjects afflictedwith tumor cells.

Experimental Details

Octahedral complexes with three bidentate ligands like phenanthroline donot cortain an inversion center, and therefore, as shown below, twoenantiomeric forms are present. Note that the intercalating portion ofthe molecule, the phenanthroline ligand, is coordinated directly to theasymmetric center of the cation, the metal. Because this chiral metalcenter is really proximal to the site of intercalation, the interactionof these complexes with DNA provides a clear illustration ofstereospecific drug binding to a similarly asymmetric DNA helix.Furthermore this stereospecific binding mode provides a means fordesigning probes for DNA helicity. ##STR1## Ruthenium(II) complexes havebeen found useful because of (i) the kinetically inert character of thelow-spin d⁶ species, (ii) their intense metal to ligand charge-transfer(MLCT) band in the visible spectrum and since (iii) many chemical andspectroscopic properties of the poly(pyridine) complexes have beenestablished. The electronic structure of the ground and excited statesof tris(bipyridine)ruthenium(II) has been examined in detail.(45) Thestrong visible absorption band, distinct from the absorption of DNA, in(phen)₃ Ru²⁺ as well as its high luminescence provide spectroscopictools to monitor the intercalative process.(38,46) Most importantly, incontrast to (phen)₃ Zn²⁺, which is somewhat labile,(47) theruthenium(II) complexes are essentially inert to racemization.(48)

Optical isomers of (phen)₃ Ru²⁺ may be isolated in pure form, (48,49)and the absolute configurations have been assigned (50). Electricdichroism measurements of (phen)₃ Ru²⁺ bound to DNA have been conducted(51) and support the findings of enantiomeric selectivity.

Although a preference in binding is found between enantiomers in thephenanthroline series, both isomers do in fact intercalate into theright-handed helix as discussed above. To amplify the chiraldiscrimination and hence improve the sensitivity of the chiral probe,suitable substituents, e.g. amino-, ethylenediamino-, hydroxyl-, nitro-,phenyl- or spermine- substituents, may be added to each phenanthrolineligand at appropriate ring sites, e.g. 3,8- or 4,7. Bulky substituentsat the distal sites on the cation can block completely the intercalationof the isomer into a right-handed helix, and thus provides selectivespectroscopic probes for the handedness of the DNA duplex. The structureof the left-handed enantiomer of a preferred complex,Λ-tris(4,7-diphenyl-1,10-phenanthroline)rutheniu m(II) (RuDIP), whichbinds to left-handed Z-DNA but not to right-handed B-DNA, is shownbelow. ##STR2## As for the smaller tris (phenanthroline) metalcomplexes, DNA binding is evident in the presence of racemic RuDIP.(52)The similarity to (phen)₃ Ru²⁺ in spectral characteristics on binding toDNA, i.e. hypochromicty and luminescence enhancements, suggested thatRuDIP also binds to the duplex by intercalation. RuDIP is howeversubstantially more bulky and hydrophobic than the parent (phen)₃ Ru²⁺cation Substitution of phenyl groups at the distal 4- and 7- positionsleads to several significant perturbations. First, the solubility of theruthenium complex in aqueous solution is diminished appreciably, whichis a practical consideration. Second, stacking with the base pairs inthe helix now requires that the phenyl groups rotate into the plane ofthe phenanthroline The rotation to planarity can be accomplished withminimal steric interactions of the neighboring hydrogen atoms bylengthening the carboncarbon bond between the phenyl and phenanthrolinemoieties; precedence for this type of structural distortion is found inthe case of biphenyl which is planar when stacked in a solid statelattice. (53) The phenyl groups once rotated into the plane of thephenanthroline increase the surface area for base pair overlapsubstantially as compared with (phen)₃ Ru²⁺, stabilizing theintercalatively bound metal-DNA complex. An increased affinity for thehelix is indeed apparent in gel electrophoresis experiments wherechanges in supercoiled DNA mobility are first evident at an order ofmagnitude lower total ruthenium concentration than for (phen)₃ Ru²⁺.Hydrophobic interactions of the non-intercalated ligands abutting thehelical groove may also account for some increase in affinity for theduplex. Third, and perhaps most importantly, the added phenylsubstituents increase the chiral discrimination markedly. While thedelta isomer can still bind closely into the right-handed helix,intercalation of the lambda isomer is now completely blocked.

Because the steric constraints are governed by the helicity of theduplex, RuDIP enantiomers can offer a specific chemical probe todistinguish right-handed and left-handed DNAs. While for the lambdaisomer steric interactions between the non-intercalated phenyl groupsand the DNA-phosphate backbone prevent close association to B-DNA, nosimilar repulsive interactions limit binding to the left-handedZ-DNA.(16) Hence an assay of duplex binding by the ruthenium enantiomersequivalently assays the DNA conformation. It is necessary at this pointto note that RuDIP is indeed a probe for helical structure and does notitself promote a conformatioion transition between B- and Z-DNA. Thecircular dichroic spectra of poly(dGC) in the B-form or Z-form withoutruthenium present are identical to those of DNA solutions containingruthenium at an added ratio of 0.05 per nucleotide. Unlike other smallerintercalators which in binding at high drug/DNA ratios cause Z--Bconformational transitions (54,55,56), racemic RuDIP does notinterconvert the helical forms. The enantiomers of RuDIP provide,therefore, a chemical means to examine DNA conformation andspecifically, the handedness of the helix. DNAs of particular repetitivesequence or those covalently bound by particular drugs will beintriguing to study. Because of the high luminescence of the rutheniumcomplexes, some interesting applications become feasible. FIG. 1 shows,for example, fluorescent micrographs(57) of Drosophila polytenechromosomes stained with racemic RuDIP. These samples were prepared incollaboration with Dr. 0.J. Miller and Dr. D. Miller. Centers of highRuDIP concentration correlate closely with the band regions seen byphase contrast and no staining is evident in either interband regions orpuffs. Experiments using the individual enantiomers can be conducted todetermine whether a high local concentration of a particularconformation is present. These micrographs should provide a usefuladdition to those obtained through staining with fluorescent antibodies,(31) with the particular advantage of pointing out specific structuralfeatures depending upon the stereoselectivity observed.

Metallointercalation reagents also offer flexibility in the design of astereospecific DNA nicking agent. Such agents have been prepared byusing tris-phenanthroline complexes with a suitable choice ofredox-active metal, e.g. cobalt. Tris(phenanthroline)cobalt(III) (58),for example, at low concentrations cleaves DNA when irradiated at 254nm. Furthermore the high stereospecificity of thetris(diphenylphenanthroline) (DIP) metal isomers (59) with DNA helicesis preserved in these cleavage reactions.

Numerous spectroscopic and x-ray crystallographic studies have shownthat DNA may adopt a range of conformations, from the right-handed A-and B-forms to the striking left-handed Z-DNA helix (60,61). Regions ofconformational heterogeneity along the strand, such as cruciformstructures, single-stranded loops, and left-handed segments, have beendetected using DNA enzymes (62), and it has been suggested that localDNA conformation might play some role in regulating gene expression.Chiral metal complexes of this invention can intercalate into the helixand are therefore particularly advantageous in probing local DNAconformation (15,34,52,64). Tris(diphenylphenanthroline) (DIP)ruthenium(II) complexes provide a spectroscopic probe for helixhandedness; the lambda isomer, which does not bind B-DNA owing to stericconstraints, binds avidly to Z-DNA (63). Upon photoactivation, theanalogous cobalt isomers, Co(DIP)₃ ³⁺, furthermore cleave DNAstereospecifically, providing a sensitive assay, for local regions inthe Z-form (64). The specific left-handed sites have now been mappedwhere Λ-Co(DIP)₃ ³⁺ cleaves in the plasmids pLP32 (29), containing ad(CG)₃₂ insert, and pBR322 (65). In pLP32 a primary cleavage cccurs atthe insert, and in native pBR322 cleavage occurs at four discrete sites:1.45 kb, 2.3 kb, 3.3 kb, and 4.2 kb. These sites correspond to segmentsof alternating purine-pyrimidines. Moreover, these positions map to theends of the three distinct coding regions in pBR322: the tetracyclineresistance gene, the origin of replication, and either end of theampicillin resistance (β-lactamase) gene. The locations of theseleft-handed segments suggest that Z-DNA might serve as a conformationalpunctuation mark to demarcate the ends of genes.

Λ-Co(DIP)₃ ³⁺, the photoactivated DNA cleaving agent used here to detectZ-DNA segments is shown below. ##STR3## The possible application of thecomplexes of this invention in chemotherapy has also been investigated.Beyond the antimicrobial and antitumor activities common tointercalating agents, if Z-DNA is important in gene regulation, thestereospecific intercalators of this invention could display a uniquepotency in vivo. Preliminary experiments in tissue cultures have shownhigh toxicity without irradiation, and phototherapy may provide greaterpotency and tissue specificity.

EXPERIMENTS Materials and Methods

I. Tris(phenanthroline)ruthenium II

Ruthenium Complexes. [(phen)₃ Ru]Cl₂ :2H₂ O was prepared as follows: toa solution of 0.5 g K₂ RuCl₅ in 50 ml hot water containing 1 drop 6 NHCl was added 0.81 g phenanthroline monohydrate (Aldrich). The resultingmixture was boiled for 15 minutes to fully dissolve the ligand andthereafter 0.75 ml of 50% hypophosphorous acid neutralized with 2N NaOHwas added. The solution was refluxed for 30 minutes and filtered hot toremove solid material. To the filtrate was added 10 ml 6N HCl, thevolume reduced to about 20 ml and cooled at 0° C. overnight. Singleorange crystals of [(phen)₃ Ru]Cl₂ were obtained. See also (38).Enantiomers were obtained by successive diastereomericrecrystallizations with antimony D-tartrate anion.(49) At most, tworecrystallizations were required to achieve [α]_(D) 1317, after whichpoint additional purification did not yield increased optical activity.Several samples of the Δ and Λ isomers were used in the course of thevarious binding studies, and, on the basis of comparison to literaturevalues for the specific rotation, all showed a level of optical puritygiven by [(C(Δ-Ru)-C(Λ-Ru)/(C(Δ-Ru)+C(Λ-Ru))]≦.92. The steroisomers wereisolated as perchlorate salts; and elemental analyses (performed byGalbraith Lab., TN) were as follows: %C, 49.34; %H, 3.29; N, 9.52;calculated for [(phen)₃ Ru] (CIO₄)₂.2H₂ O, %C, 49.32; %H, 3.22; %N,9.59. Spectrophotometric and luminescence titrations of racemicRu(phen)₃ Cl₂ and equimolar mixtures of Δ- and Λ-Ru(phen)₃ (CIO₄) withDNA agreed closely, indicating that the presence of perchlorate (≦50 M)was without effect. Stock ruthenium solutions were either freshlyprepared or kept in the dark. Concentrations of (phen)₃ Ru²⁺ weredetermined spectrophotometrically by using ε₄₄₇ 1900 M⁻¹ cm¹.(38)

BUFFERS AND CHEMICALS. Experiments were carried out at pH 7.1 in buffer1 (5 mM Tris, 50 mM NaCl), buffer 2(5 mM Tris, 4.0 M NaCl), or buffer 3(50 mM Tris acetate, 20 mM sodium acetate, 18 mM NaCl pH 7.0). Solutionswere prepared with distilled deionized water. Plasticware was usedthroughout and was cleaned by soaking overnight in 10% HNO₃ followed byexhaustive rinsing. Dialysis membranes were prepared by the followingprotocol: After they were boiled successively in sodium carbonate, 1%EDTA, and 1% SDS and exhaustively rinsed in deionized water, themembranes were heated to 80° C. in 0.3% sodium sulfite, acidified at 60°C. with 2% sulfuric acid, and thereafter rinsed again with deionizedwater and 1% EDTA. This procedure serves to minimize metal binding tothe membranes.

Nucleic Acid. Calf thyxus DNA, obtained from Sigma Chemical Co., waspurified by phenol extraction as described previously.(9c)Poly(dGC).poly(dGC) from P.L. Biochemicals Inc. and plasmid ColEl fromSigma Chemical Co. were extensively dialyzed in buffer before use. DNAconcentrations per nucleotide were determined spectrophotometrically byassuming ε₂₆₀ 6000 M⁻¹ cm⁻¹ for calf thymus DNA and ε₂₆₀ 8400 M⁻¹ cm⁻¹for poly(dGC).

Spectroscopic Measurements. Absorption spectra were recorded on a Cary219 spectrophotometer. Absorbance titrations of racemic, Δ-and Λ-(phen)₃Ru²⁺ in buffer 1 were performed by using a fixed ruthenium concentrationto which increments of the DNA stock solution were added. Ruthenium wasalso added to the DNA stock to keep the total dye concentrationconstant. Luminesence measurements were conducted on a Perkin-Elmer LS-5flourescence spectrophotoxeter at ambient temperature. Samples wereexcited at 447 nm, and emission was observed between 500 and 700 nm. Allexperiments were carried out in buffer 1 with (phen)₃ Ru²⁺concentrations typically of 10 μM and DNA phosphate/ruthenium ratiosvarying from 1 to 50. Lifetime measurements were performed on an Ortec776 single-photon counter and timer in line with an Apple Computer. Thesamples were excited with a PRA 510A nanosecond lamp, and emission wasobserved at 593 nm. Reproducible lifetimes for the bound species in thepresence of free ruthenium were obtained by neglecting the first 1.2μs(2τ[Ru(phen)₃ ²⁺ _(free), of the decay curve.

Electrophoresis. Dye gel electrophoresis of supercoiled DNA in 1%agarose was performed in buffer 3 by using the method of Espejo andLebowitz (6) modified as described previously.(15) Rutheniumconcentrations in the gels were carefully determined on the basis ofseveral absorbance readings of the stock concentrations for theenantiomers. Because of the high background luminescence of (phen)₃Ru²⁺, gels were destained for 24 hours in buffer prior to staining withethidium.

Equilibrium Dialysis. Binding isotherms were obtained on the basis ofdialysis of calf thymus DNA in buffer 1 against (phen)₃ Ru²⁺ at 22° C.The DNA was dialyzed first exhaustively in buffer to remove smallfragments. Thereafter, dialysis against ruthenium was allowed tocontinue for at least 24 hours after which time equilibration wasachieved. Each sample consisted of 2mL of dialysate containing Δ-,Λ-, orrac-(phen)₃ Ru²⁺, varying in concentration between 50 and 1,000 μM, and,within the dialysis bag, 1 mL of 1 mM DNA phosphate. To determine boundand free concentrations, absorbance spectra were taken of dilutions(3-50 μM). Free ruthenium concentrations outside the bag were determinedon the basis of absorbance readings at 447 nm. For concentrations ofruthenium inside the bag, in the presence of DNA, readings were obtainedat the isosbestic point, where ε₄₆₄ 13630 M⁻¹ cm⁻¹ (vide infra).Equilibrium dialysis of poly (dGC) was conducted similarly in buffer 2.Measurements of circular dichroism were obtained on a Jasco J-40automatic recording spectropolarimeter. Because of the irregularbaseline of the instrument, all spectra were digitized and replottedafter base-line subtraction. Data analyses were performed on a IBM PCand a Digital VAX 11/780.

II. Tris(4,7-diphenyl-1,10-phenanthroline)rutheniumII

Nucleic Acids. Calf thymus DNA Sigma) was purified by phenol extraction(66). Poly[d(G-C)] (P-L Biochemicals) was dialyzed at least three timesbefore use. Experiments were conducted at pH 7.2 in buffer 1 [4.5 mMTris.HCl/45 mM NaCl/150 μM Co(NH₃)₆ Cl₃ /10% dimethyl sulfoxide], buffer2 [5 mM Tris.HCl/50 mM NaCl/150 μM Co(NH₃)₆ Cl₃ ], or buffer 3 [5 mMTris.HCl/4.0 M NaCl]. DNA concentrations per nucleotide were determinedspectrophotometrically assuming ε₂₆₀ =6600 M⁻¹.cm⁻¹ for calf thymus DNA(67) and ε₂₆₀ =8400 M⁻¹.cm¹ for poly[d(G-C)] (68). In preparing Z-DNA,poly[d(G-C)] stock solutions weree incubated in the cobalt hexamminebuffer for 2-18 hr to ensure both a complete transition to the Zconformation and minimal aggregation. Stock solutions were examinedspectrophotometrically and by CD before use.

Ruthenium Complexes. The synthesis of RuDIP trihydrate was carried outas described above, substituting 4,7-diphenyl-1,10-phenanthroline forthe unsubstituted 1,10-phenanthroline. See also (38). Concentrationswere determined spectrophotometrically using ε₄₆₀ =2.95×10⁴ M⁻¹. cm⁻¹.Elemental analyses were consistent with literature values. The Δ and Λisomers were either separated by successive recrystallizations with theantimony tartrate anion in 50% ethanol or prepared by asymmetricsynthesis in the presence of antimony tartrate and then recrystallized.Λ-RuDIP forms the less soluble diastereomeric salt with antimonylD-tartrate. The separated isomers were isolated finally as perchloratesalts. The assignments of absolute configuration have been made on thebasis of the relative binding affinities of these enantiomers for B-DNA(see below). Many rounds of recrystallization yielded a small quantityof Λ-RuDIP having [θ]₂₈₃ =-4.0×10³ deg. M⁻¹. cm. This assignment isconsistent with both the UV CD fortris(1,10-phenanthroline)ruthenium(II) [(phen)₃ Ru²⁺ ], assignedpreviously (31), and studies of the enantiomeric preference of (phen)₃Ru²⁺ for B-DNA (34, 35, 51). The optical purities of the Δ- and Λ-RuDIPsamples used below were 41% and 70%, respectively. Therefore the sampledesignated Δ-RuDIP contains 70.5% Δ isomer and 29.5% Λ isomer, and thatdesignated Λ-RuDIP is composed of 14% Δ- and 86% Λ-RuDIP.

Spectroscopic Measurements. Atsorbance spectra were recorded using aVarian Cary 219 UV/visible spectrophotometer and luminescence spectra,with a Perkin-Elmer LS-5 fluorescence spectrophotometer. Titrations werecarried out using a constant ruthenium concentration (4-6 μM) to whichincrements of either calf thymus DNA or solubility in aqueous solution(≦10 μM), dimethyl sulfoxide was included in buffer 1. CD spectra ofB-DNA or Z-poly[d(G-C)] with 150 μM Co(NH₃)₆ ³⁺ were unaffected by thepresence of the dimethyl sulfoxide. Although more difficult, titrationsin buffer 2 and buffer 3 were also conducted.

III. CobaltIII Complexes

Tris(4,7-diphenyl-1,10-phenanthroline)cobalt(III) (Co(DIP)₃ ³⁺)tri-tartrate was prepared as follows: 4,7-diphenyl-1,10-phenanthroline(Aldrich) was dissolved in a minimum volume of ethanol to which onethird stoichiometric CoCl₂.6H₂ O was added. The green brown solution wasoxidized by using Br₂ /H₂ O, and a heavy orange precipitate formedimmediately. The solution was refluxed for 1 h, and concentratedhydrochloride was added. The bromine oxidation was then repeated. Thecrude chloride salt was used directly for enantiomeric separations. Witheither 1- or d-tartaric acid (Aldrich), the deep red tartrate (Tar)diastereomeric salts [Λ-Co(DIP)₃ ].(L-Tar)₃ and [Δ-Co(DIP)₃.(d-Tar)₃,were prepared by successive recrystallizations in 50% ethanol, pH 7.0.

Chemical and spectroscopic data for these complexes are as follows:Anal. Calcd for [Co(DIP)₃ ](Tar)₃.H₂ O (CoC₈₄ N₆ O₁₉ H₆₅) C, 66.32; H,4.32; N, 5.52; Found: C, 65.87; H, 4.46; N, 5.78. Absorption spectrashowed λ_(max) at 278 and 312 nm (shoulder). The circular dichroicspectra resemble those of enantiomers of Ru(DIP)₃ ²⁺, and absoluteconfigurations have been assigned on that basis.

IV. Cleavage Methods [Co(DIP)₃ ](tartrate)₃ (10 μM) was added to pBR322DNA (100 μM nucleotides) in 50 mM tris-acetate buffer containing 18 mMNaCl, ph 7.0. The 20 μl sample was then irradiated at 315 nm (with a1000 W Hg/xenon lamp narrowed to 315±5 nm with a monochrometer) for 90seconds and ethanol precipitated. The ethanol wash removes unreactedCo(DIP)₃ ³⁺ as well as the metal and ligand products of the reaction.After resuspension in trisacetate buffer containing 50 mM NaCl and 10 mMMgCl₂, pH 7.0, restriction enzyme was added (either EcoRI, BamHI, AvaIor NdeI) using at least a threefold excess to insure completelinearization. This was incubated at 37° C. for 45 min. The pH of thereaction mixture was then lowered to 5.0 and 10 mM Zn(NO₃)₂ added alongwith 4 units of S1 nuclease, and the samples were incubated for 5 min at37° C. This step causes cleavage of the DNA by S1 opposite the sitenicked by Co(DIP)₃ ³⁺. Electrophoresis on 1% agarose gels followed (50mM tris-acetate, 18 mM NaCl, pH 7.0) to resolve the double strandedfragments produced. In these experiments pBR322 sequences are numberedbeginning at the EcoRI site according to Sutcliffe (65). Gels werestained with 5 μg/ml ethidium bromide for 0.5 hr then destained inbuffer for 2 hr. Gels were photographed using a Polaroid 600 camera witha red filter and 615 positive/negative film and irradiated from below.

V. In vitro Screening

For cell culture studies, a modification of the techniques of Fischer(69) was used. The cells were incubated in McCoy's Medium 5A with 15%fetal calf serum. The initial inoculum was 40,000 to 60,000 leukemiccells/ml. For studies of the inhibition of cell growth, 0.1 ml of a20-fold concentraticn of the drug in question was added to 2 ml of mediacontaining 4×10⁴ cells/ml in Linbro tissue culture multiwell plates andallowed to incubate at 37° in 5% CO₂ for 96 hr. By these times, growthto approximately 10⁶ cells/ml occurred in the control wells. Thecontents of each well were agitated to resuspend the cells and countedon a Coulter Counter. The percentage of inhibition of growth and theconcentrations inhibiting cell growth by 50% were calculated. Cellculture experiments were conducted with mouse leukemia cell lines L1210and P815. The cell lines and growth medium may be obtained from theAmerican Type Culture Collection (ATCC), Rockville, Md.

Results

I. Tris(1,10-phenanthroline)ruthenium (II)

Spectroscopic Studies

The binding of Λ- and Δ-(phen)₃ Ru²⁺ to duplex DNa leads to a decreaseand small shift in the visible absorption of the ruthenium species and acorresponding increase and shift in luminescence. FIG. 2 shows both theabsorption spectra and luminescence spectra of (phen)₃ Ru²⁺ in thepresence and absence of calf thymus DNA. The spectral changes observedhere are often characteristic of intercalation.

The hypochromic shift in the broad charge-transfer band of (phen)₃ Ru²⁺as a result of binding to the polynucleotide can be seen in FIG. 2A. Adecrease of 12% in absorbance at 447 nm is found for the saturating DNAlevels employed. Since at these concentrations 70% (phen)₃ Ru²⁺ is inthe bound form, ε(bound)/ε(free)=0.83 at 447 nm. The hypochromic effectis small compared with that found for other intercalators, which mayindicate that the charge is not being preferentially localized onto theintercalated ligand. Also for the free ruthenium complex the predominantpolarization of the charge-transfer band is perpendicular to themolecular C3 axis (46) rather than parallel to the intercalative plane.Shown in the figure is the change in absorbance for the racemic mixture;also because the observable hypochromic effect is small, significantdifferences between enantiomers were not obtained. For both isomers aspectral shift of 2 rm to lower energy is found, which supports anelectronic stacking interaction of the phenanthroline ligand with thebase pairs of the helix. Isosbestic points at 355 and 464 nm are alsoapparent.

An enhancement in the luminescence of (phen)₃ Ru²⁺ on binding to duplexDNA parallels the observed hypochromicity. FIG. 2B shows the emissionspectra of free (phen)₃ Ru²⁺ and of both lambda and delta isomers boundto DNA. These spectra also reveal a shift of 2 nm to longer wavelengthwith DNA binding. Moreover, in the presence of 0.25 mM DNA phosphate,emission increases of 48% and 87% are observed respectively for Λ- andΔ-(phen)₃ Ru²⁺ (10 μM). Note that a significant fraction of therutheniux is free in the presence of the DNA at these concentrations,but the associated increase in solution viscosity for higher DNAconcentrations precluded studies at saturating binding levels. Thegreater increase in luminescence seen for the Δ isomer in the presenceof DNA over that for the Λ isomer indicates simply that a higherproportion of the Δ isomer is bound, rather than that their modes ofassociation with the helix differ. Measurements of the excited-statelifetimes of enantiomers in the absence and presence of the DNA yieldedresults consistent with this interpretation. For Δ- and Λ-(phen)₃ Ru²⁺,determined separately and as a racemic mixture, identical experimentallifetimes of 2.0 and 0.6 μs were found respectively in the presence andabsence of DNA. Both isomers therefore bind to the helix in a similarfashion, and indeed, if fully bound, would display similar enhancementsin luminescence. Substantial increases in fluorescent lifetimes withintercalation have been observed in several instances (2-4,70), notablyfor ethidium, and may be explained by the greater rigidity and lowercollisional frequency of the molecule when stacked within the helix.

Measurements of Helical Unwinding

Both Λ- and Δ-(phen)₃ Ru²⁺ reversibly unwind and rewind supercoiled DNAas a function of increasing concentration of ruthenium(II), and for agiven total concentration, a greater unwinding effect is evident for theΔ isomer. FIG. 3 illustrates the migration of pColEl DNA through 1%agarose gels containing increasing levels of (phen)₃ Ru²⁺. Mobilitiesare plotted relative to the supercoiled DNA control to permit theinclusion of data from several gel electrophoresis trials. As can beseen in the figure, both isomers unwind the helix. With increasinglevels of ruthenium bound, the duplex unwinds, and for a closed circlethis unwinding results in first a release of negative supercoils at lowlevels bound and then the introduction of positive supercoils; thenicked DNA, without similar topological constraints, displays novariation in mobility. The bars in the figure indicate the width of theDNA bands, which vary because of the distribution of topoisomers in thesample. The observed duplex unwinding provides a strong indication ofintercalative binding. Control experiments also show the unwinding to bereversible; preincubation of the DNA with ruthenium complex has noeffect on gel mobility. It is interesting to note that no DNA cleavageis observed as a result of binding (phen)₃ Ru²⁺, even after irradiationwith ultraviolet light (short wavelength) for 1 h.

For a given level of total ruthenium, a higher amount of duplexunwinding is found in the presence of Δ-(phen)₃ Ru²⁺, respectively. Thecomigration of nicked and closed circular DNAs occurs in the presence of90 and 120 μM Δ-(phen)₃ Ru²⁺. This comigration point represents a fixedamount of helical unwinding. A lower added concentration of Δ-(phen)₃Ru²⁺ is needed to unwind all the negative supercoils in the pColEl DNA.These results therefore also reflect the higher affinity of the Δ isomerover the A isomer for the right-handed helix. At a given totalconcentration of ruthenium, more of the Δ isomer is bound and greaterhelical unwinding is evident. The alternative explanation for the lowerconcentration of the Δ isomer at the comigration point would be that theΔ isomer has a larger unwinding angle than the Λ isomer, so that the Aisomer unwinds the duplex more per drug bound. A particularly largedifference between unwinding angles (about 30%) would be needed toaccount for the electrophoresis results, however, and only smallvariations in unwinding angles are generally observed (4). Moreoverlarger, if any, structural perturbations should accompany binding of theΛ isomer to the right-handed helix rather than the Δ isomer. Here then,as well, the results show that the Δ isomer possesses a greater affinityfor the DNA duplex.

These data may be used to estimate the intercalative unwinding angle. Ifwe assume for the racemic mixture that the average comigration point ofnicked and closed forms occurs with 100 μM ruthenium, then, on the basisof our determination of the binding constant (vide infra), a bindingratio of 0.089 per nucleotide is required to unwind fully the supercoilsin the plasmid. Interestingly this value is identical with thatcalculated for ethidium, since in buffer 3 the comigration of nicked andclosed pColEl species in the presence of 5×10⁻⁷ M dye was observed.Therefore the unwinding angle for (phen)₃ Ru²⁺ is estimated to be thesame as that for ethidium (6).

Equilibrium Dialysis Experiments

The results of dialysis of calf thymus DNA with racemic (phen)₃ Ru²⁺ at22° C. in buffer 1 are shown in FIG. 4 in the form of Scatchard plot(71). The data have been fit by nonlinear least-squares analysis to thefollowing equation governing noncooperative binding to the helix, asderived by McGhee and von Hippel (72): ##EQU1## where r is the ratio ofthe bound concentration of ruthenium to the concentration of DNAphospate, CF is the concentration of ruthenium free in solution, K(O) isthe intrinsic binding constant, and the integer I, which measures thedegree of anticooperativity, is the size of a binding site in basepairs. In fitting the data, the binding parameter K(O) was varied forseveral integer values of L. The best fit, shown as the solid curve inFIG. 4, yielded a binding constant K(O)=6.2×10³ M⁻¹ (±2%) and anexclusion site size (L) of four base pairs. Data from luminescencetitrations were consistent with this curve. The binding constant isquite low in comparison to values of 3×10⁵ and 5×10⁴ M⁻¹ (extrapolatedto the ionic strength of our buffer) for ethidium and [(phen)Pt-(en)]²⁺,respectively (73). The lower affinity of (phen)₃ Ru²⁺ is not surprisingsince only partial stacking of the phenanthroline ligand is feasible inthis octahedral complex; greater overlap of the phenanthroline with thebase pairs may be achieved in the square-planar platinum(II) species.The steric bulk of the nonintercalated ligands determines also the largefour base-pair site size compared to a two base-pair (neighbor excluded)site for basically planar reagents (9,74). Inspection of space-fillingmodels show that the perpendicular phenanthroline ligands each span twobase pairs either above or below the intercalatively boundphenanthroline, which is consistent with the binding isotherm.

In these equilibrium dialysis experiments of the racemic mixture, therelative binding of the two enantiomers to the polynucleotide may bedetermined sensitively on the basis of the degree of optical enrichmentof the unbound enantiomer in the dialysate. FIG. 5 shows the circulardichroism (1.5×10⁵ M) of the dialysate after equilibration of calfthymus DNA (1 mM) with racemic (phen)₃ Ru²⁺ (2×10⁻⁴ M) Also shown forcomparison is the circular dichroism of Δ-(phen)₃ Ru²⁺ (0.2 μM). Thespectra show clearly that the dialysate has been optically enriched inthe less favored isomer. The Δ enantiomer binds preferentially to theright-handed helix. The degree of chiral discrimination may be made morequantitative by comparing the level of optical enrichment (2% for thesample shown) with the overall amount of ruthenium bound. On the basisof a simple competition between the enantiomers for sites along thehelix with no cooperativity and, for simplification, describing thebinding by each enantiomer in terms of the familiar Scatchard equation,X, the ratio of binding constants K(Δ)/K(Λ ) may be calculated asfollows: ##EQU2## where P is the concentration of DNA phosphate, n isthe ratio of drug to DNA phosphate bound at saturation, taken as 0.125,C_(B) is the total concentration of ruthenium bound, and ΔC is thedifference in free concentrations between Δ and Λ isomers as measured bythe intensity in the circular dichroism. Measurements of several samplesyielded values of 1.1-1.3 for X. Thus the binding affinity of Δ-(phen)₃Ru²⁺ is found to be 10-30% greater than Λ-(phen)₃ Ru²⁺ for calf thymusDNA by this method. This value is comparable to the differences seen inluminescence and unwinding experiments. Since the overall binding of(phen)₃ Ru²⁺ is small, binding isotherms obtained through equilibriumdialysis tended to show some scatter. A direct comparison of the bindingisotherms for the enantiomers in equilibrium dialysis experiments usingthe pure isomers therefore could not be achieved; significantdifferences were not evident. Interestingly it appears that the methodof optical enrichment yields the most sensitive assay for thedifferential binding.

Since the enrichment experiment provides the most sensitive method toexamine enantiometric discrimination, poly(dGC) in 4M NaCl was alsodialyzed against rac(phen)₃ Ru²⁺ to test for any enantiomericpreferences in binding to a left-handed DNA helix (16,17). At the lowbinding levels examined, the circular dichroism of the polymer remainsinverted, indicating that racemic (phen)₃ Ru²⁺ did not induce a Z-- Btransition. After equilibrium dialysis with bound concentrationscomparable to those in earlier experiments using calf thymus DNA, e.g.,under conditions where low levels of enrichment could be detected, nooptical activity was observed in the dialysate. Therefore, althoughintercalative binding had occurred, given similar spectralcharacteristics as in binding to the right-handed helix, no preferencein binding was evident. In FIG. 5 the essentially base line spectrum ofa solution after dialysis against Z-form poly(dGC) has also beenincluded. This lack of discrimination for (phen)₃ Ru²⁺ is understandablein view of the shallow, almost grooveless character of the left-handedZ-DNA helix.

II. Tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II)

In spectroscopic studies of raremic RuDIP with B- and Z-DNA, changes areseen in both the visible absorption and luminescence spectra of RuDIP onaddition of either B- or Z-form DNA. Hence, binding may be monitoredsensitively using either spectroscopic technique. Visible absorptiontitrations of racemic RuDIP in buffer 1 (described below) with calfthymus DNA and Z-form poly[d(G-C)] are shown in FIG. 6. The overallsimilarity of these titrations is apparent. Binding of either duplex DNAleads to hypochromicity in the intense metal-to-ligand charge-transferband of the ruthenium complex. A small red shift (about 2 nm) in thespectrum of the bound complex and an isosbestic point at 485 nm can beseen. That spectral changes occur as a function of addition of eitherDNA form suggests that racemic RuDIP binds to both B- and Z-DNA. Thesimilarity in spectral changes most likely reflects a similar mode ofassociation of the ruthenium complex with either the right-handed B-DNAhelix or the left-handed Z-DNA helix.

Differences in binding to the two forms are evident, however. A greaterreduction in the absorption intensity of Ru-DIP accompanies binding toZ-form poly[d(G-C)] than to the B-DNA helix. In FIG. 6, for example, theapparent reduction in intensity with the addition of a 13:1 ration ofcalf thymus DNA-phosphate/ruthenium is only 9% whereas, for theleft-handed helix, the reduction occurring at a nucleotide/rutheniumratio of 5:1 is 17%. The greater hypochromicity in binding to Z-DNA isexplained in part by the different stereoselectivities governing bindingto each helix. Although both enantiomers bind to Z-DNA, only the Δenantiomer may bind easily to right-handed B-DNA. The differences instereoselectivity cannot fully account for the difference inhypochromicity, however, because the hypochromicity in spectra ofracemic RuDIP with Z-DNA is more than twice that observed with calfthymus DNA. If one assumes that the extinction coefficients for RuDIPwhen bound to each helix are the same, which seems reasonable based onthe equal isosbestic prints observed, then the larger hypochromic effectwith Z-DNA suggests that both RuDIP enantiomers possess a greateraffinity for Z-form poly[d(G-C)] than for calf thymus DNA. Equilibriumdialysis experiments support this conclusion.

The luminescence of RuDIP is also enhanced on binding to the DNA duplex.FIG. 7 shows the emission spectrum of racemic RuDIP (3 μM) in theabsence and presence of calf thymus DNA and Z-form poly[d(G-C)] (15 μMnucleotide). The shift in the spectrum to lower energy is particularlypronounced despite the broad nature of the transition; the maximumshifts 10 nm to longer wavelength in the presence of DNA. Greaterluminescence is seen here on binding to B-DNA, despite the lowerapparent affinity for this helix. In buffer 2, at aDNA-phosphate/ruthenium ratio of 5:1, the emission intensity of racemicRuDIP increases by 30% and 47% in the presence of Z-form poly[d(G-C)]and calf thymus DNA, respectively. The different enhancements may dependin part on nucleic acid composition as well as duplex conformation,RuDIP bound to B-form poly[d(G-C) yields less luminescence than whenbound to calf thymus DNA, despite having an equal affinity for thesepolynucleotides.

Racemic RuDIP appears to bind to both B- and Z-DNA rather than promotinga transition from one conformation to the other. CD spectra of Z-formpoly[d(G-C)] in Co(NH₃)³⁺ (buffer 1) or in 4 M NaCl (buffer 3) areunaltered by the addition of racemic RuDIP at a nucleotide/rutheniumratio of 10. Conversion of the Z-form to B-form with RuDIP isinconsistent also with the differential hypochromism and luminescenceobserved. If RuDIP promoted a Z to B transition, albeit inefficiently,rather than binding to both B- and Z-helices, then both the reduction inabsorbance and the enhancement in luminescence observed on addition ofZ-DNA would be less than or equal to that found with B-DNA, i.e., inproportion to the fraction of DNA converted. Instead significantlygreater hypochromism is found when Z-DNA rather than B-DNA is added tothe racemic mixture or indeed to each enantiomer individually. Thereforeracemic RuDIP must bind to both DNA comformations Consistent with theseresults, a conformational transition from Z-DNA to B-DNA would not beexpected if the affinity of the metal cation for the Z-form were greaterthan that for the B-form. The ethidium cation, which binds to B-DNA byintercalation, is known to promote a Z to B transition at high bindingratios (75,76) and presumably possesses a greater affinity for theB-form helix. The substantially larger RuDIP cation cannot saturate theDNA to comparable levels, which may be an important distinction.Moreover, although the ethidium ion can fully intercalate into B-DNA,RuDIP cannot and the nonintercalating ligands of RuDIP x-ray dominateits interactions with the duplex.

The utility of the RuDIP enantiomers as a probe for helical conformationbecomes apparent when the binding characteristics of each enantiomer toB- and Z-DNAs are compared. Plots of the relative absorbance at 460 nmof the individual enantiomers as a function of the addition of eitherB-form calf thymus DNA or Z-form poly[d(G-C)] in buffer 1 are shown inFIG. 8. Based on the presence or absence of hypochromicity, it is clearthat although RuDIP binds to B-DNA, the Λ isomer does not. ΔRuDIP doeshowever bind to Z-DNA. Indeed, with Z-DNA no stereospecificity isobserved Hence the assay of duplex binding by the Λ isomer yields asensitive assay for the Z-DNA conformation.

Strong enantiomeric selectivity governs the interaction of RuDIP withthe right-handed B-DNA helix. The decrease in absorbance with increasingDNA concentration observed for the Λ, racemic mixture, and Δ samples canbe fully accounted for based on the percentage of the Δ enantiomerpresent in the particular preparation (see Experimental). The pure Λenantiomer does not bind to B-DNA. The presence of the phenyl groups atthe 4 and 7 positions of the nonintercalated phenanthroline ligand hasserved to amplify the chiral discrimination. In comparison, differencesin binding of (phen)₃ Ru²⁺ enantiomers had been seen only inspectrophotometric titrations at high DNA/ruthenium levels; the ratio ofthe affinities for B-DNA of Δ to Λ isomers is about 1.3(34,35). ForRuDIP, with hydrogen atoms now replaced by phenyl groups, instead ofsimple interference with the DNA phosphate oxygen atoms, one finds thatthe steric bulk of the phenyl groups completely blocks interactions ofthe isomer in the right-handed groove. Δ-RuDIP, however, binds withfacility to a right-handed helix, indeed more avidly than Δ-(phen)₃Ru²⁺. This striking amplification in enantiomeric selectivity for RuDIPcompared with (phen)₃ Ru²⁺ strongly supports our model forstereospecific intercalation

Z-DNA serves as a poor template to discriminate between the enantiomers;identical reductions in absorbance intensity are found for the Δ and Λisomers (FIG. 8). Because of the shallow and very wide character of themajor groove in Z-DNA, there are no steric constraints comparable withthat found with B-DNA. Hence, if the binding modes are equivalent, nochiral specificity would be expected. The similarity in spectralcharacteristics of RuDIP in binding to the different DNA duplexes pointsto this similarity in binding modes. However, the lack of chiralspecificity in binding to Z-DNA does limit what car be said at presentabout the interaction of RuDIP enantiomers with a Z-form helix. Based onrelative hypochromicities, it appears that both Λ and Δ-RuDIP possessgreater affinities for Z-form poly[d(G-C)] than for B-DNA. Hydrophobicinteractions with the helical surface may lend some stability to thebound complex(77). The difference in affinity furthermore does notreflect a preference for base composition. Titrations of racemic RuDIPwith B-form poly[d(G-C)] in buffer 1 lacking cobalt hexammine showedhypochromicity equal to that seen with calf thymus DNA. Also, cobalthexammine itself does not appear to alter binding to the helix. RuDIPtitrations using calf thymus DNA with and without Co(NH₃)₆ ³⁺ wereidentical. In addition, the interaction cannot be explained purely beelectrostatic interactions. Although smaller, hypochromic effects,approximately one-third of that shown here, are found in titrations in 4M NaCl (buffer 3, with either poly[d(G-C)] or calf thymus DNA. Partialintercalation into the DNA by both RuDIP enantiomers would be consistentwith these results. It is finally important to note that the similartitrations of both enantiomers that are seen with Z-DNA but not withB-DNA suggest that neither enantiomer converts the Z-form helix to theB-DNA conformation. If that were the case, selectivity between theenantiomers would become apparent.

III. Cobalt (III) Complexes

FIG. 9 shows gel electrophoretic separations of plasmid ColEI DNA afterincubation with cobalt complexes and irradiation for variable times. DNAcleavage is followed by monitoring the conversion of supercoiled (formI) closed circular plasmid DNA to the nicked circular form (form II) andlinear (form III) species. (The original ColEl preparation contained 60%form I and 40% form II molecules.) FIG. 9A reveals the completeconversion of form I to II after a 1-h irradiation in the presence of 10M (phen)₃ Co³⁺. Neither irradiation of the DNA at these low intensitieswithout cobalt nor incubation with cobalt without light yieldedsignificant strand scission. (Irradiation at 310nm where there arestrong ligand transitions also leads to cleavage). It is likely that thereduction of Co(III) is the important step leading to DNA cleavage andnot that irradiation provides a means to generate cobalt(II) in situ.DNA incubation with the tris(phenanthroline) complex initially in thecobaltous form yielded no reaction unless irradiated. Presumably thecobaltous complex is oxidized in solution to the cobaltic species, sinceit is the +3 oxidation state in cobalt polyamine complexes that isphotochemically active. Also dithiothreitol inhibits activity ofCo(phen)₃ ³⁺, perhaps by precluding regeneration of an activecobalt(III) species. This finding is in contrast to the iron and coppersystems where thiols are thought to stimulate activity by generating themetal species in the reduced form (39-43). Interestingly,electrophoresis also reveals with increasing irradiation a smallreproducible increase in the mobility of form II; this may reflect someshort-range radical-induced DNA cross-linking (78).

The cleavage reaction is furthermore strongly stereospecific. FIG. 1Bshows pColEl DNA of low superherical density after incubation witheither Λ-Co(DIP)₃ ³⁺ or Δ-Co(DIP)₃ ³⁺ (17,18) and irradiation withultraviolet light. Incubation of pColEl DNA of low superhelical densitywith the Λ isomer, which cannot bind to a right-handed duplex owing tosteric constraints, yields no appreciable reaction (19), whereasincubation with Δ-Co(DIP)₃ ³⁺, which is able to associate closely withright-handed B-DNA, shows efficient nicking activity comparable to thatseen with Co(phen)₃ ³⁺. Nicking was observed, however, upon titration ofpColEl of increasing superhelical density with ΛCo(DIP)₃ ²⁺. Thisdifferent cleavage efficiency by each enantiomer is consistent with theearlier finding (7) of conformational discrimination by theruthenium(II) isomers; one enantiomer of Ru(DIP)₃ ²⁺ binds to B-DNA, butboth Δand Λ-Ru(DIP)₃ ²⁺ bind to the left-handed Z-DNA helix. Theseresults underscore the importance of an intimate association of themetal with the duplex.

In FIG. 9B the overall concentrations of the cobalt isomers are equal,yet the A trication, if it cannot intercalate, does not yield DNA strandscission.

The Λ-tris(phenanthroline) metal complexes do, however, bind toleft-handed Z-DNA (21,22). We examined the plasmid pBR322 containing a42-base pair alternating guanine-cytosine insert (pLP42) (29,79) andwhich was shown (28,17) to adopt the Z-conformation in 4 M NaCl. Underthese conditions, cleavage by both Co(DIP)₃ ³⁺ enantiomers is obtained.Hence the isomer may recognize and cleave left-handed helices. Moreinteresting, however, is the finding, plotted in FIG. 10 as percent lossof supercoiled form, that the plasmid pBR322 at physiological saltconcentrations and without extreme superhelix underwinding also issignificantly cleaved by the isomer. Given our other results ofdifferential binding based on DNA helicity (35,63) and the differentialcleavage of ColEl of low superhelical density described above, itappears that Λ-Co(DIP)₃ ³⁺ might bind to and cleave a naturalleft-handed segment in pBR322 DNA of low superhelical density in normalsalt concentrations. These observations support the findings by Rich andco-workers (80) of anti-Z antibody binding to the 14-base pairalternating purinepyrimidine segment in pBR322). The statisticallysignificant 14 base pair sequence (CACGGGTGCGCATG) in pBR322 showsalternation of purine and pyrimidine with one base out of register.Alternating purine-pyrimidine sequences tend to adopt the Zconformation. The plasmid pColEl sequence contains no comparable stretchof alternation.

IV. Cleavage Site Mapping

Irradiation at 315 nm of Λ-Co(DIP)₃ ³⁺ (10 μM) solutions containingsupercoiled pLP32 or pBR322 yields nicked circular form II DNAs.Photoreduction of Co(DIP)₃ ³⁺ enantiomers bound to DNA leads tooxidative single-strand scission at the DNA binding site (64). In orderto establish that cleavage and therefore binding occurs at discretesites, the scheme outlined in FIG. 11 was employed. Followingirradiation of Co(DIP)₃ ³⁺ -DNA samples and the production of nickedcircles, the DNAs were linearized using a restriction enzyme which isknown to cleave the plasmid at only one site along the strand.Subsequent treatment with S1 nuclease, which is specific forsingle-stranded regions, cleaves the DNA only opposite to thecobalt-induced nick producing a pair of linear fragments. From the sizesof these fragments, determined based upon their gel electrophoreticmobilities relative to markers, the distance of the cleavage site fromthe restriction site origin may be obtained. In order to distinguishwhether the site is clockwise or counterclockwise to the origin, atleast two restriction enzymes which cut at sufficiently distinctlocations were examined. It is important to notice that this procedureyields distinct fragments only if binding and subsequent cleavage occursat discrete sites. Non-specific cleavage produces fragments of all sizesand hence a smear on the gel; thus the presence of some contaminatingform II DNA just alters the background intensity. Control experiments ofsample irradiated without cobalt or cobalt binding but withoutirradiation yielded no distinct bands. Non-specific DNA damage as aresult of irradiation was negligible. Controls showed that fulllinearization of the plasmid was essential, however, to avoid mapping S1hypersensitive sites (62). Some restriction enzymes did not yieldcomplete linear digests, either because of thymine dimer formation atthe restriction site or inhibition due to Co(DIP)₃ ³⁺ reaction, andthese were not used. Finally, samples were irradiated only for shorttimes so that no more than one nick per plasmid would occur. The factthat the sizes of pairs of fragments must sum to 4363 base pairsprovided a useful experimental redundancy. By this general procedure thecoarse map of Λ-Co(DIP)₃ ³⁺ cleavage sites in any plasmid may beobtained.

The plasmid pLP32 which contains a Z-DNA segment at a well-definedlocation (29,81) was examined first. The plasmid had been constructed byinserting a d(CG)₃₂ fragment into the filled-in BamHI site (position375) of pBR322 (29). The densitometric scan of the AvaI digest afterreaction with Λ-Co(DIP)₃ ³⁺ is shown in FIG. 12. In addition to thelinear form several bands and shoulders are evident; their sizes inkilobase pairs (kb) are indicated in the figure. The appearance of thepair of fragments at 3.3 kb and 1.1 kb from the AvaI site (position1424) shows that a major cleavage point is indeed at the Z site.Parallel digestion with Ndel established this position uniquely Moreinteresting perhaps is the comparison to the AvaI digest of pBR322, thesame plasmid but lacking the insert. The pattern here is identicalexcept that it lacks the 3.3 kb and 1.1 kb fragments. In pLP32, then,the cobalt complex must recognize and cleave a site not present inpBR322, the Z-form d(CG)₃₂ insert. The result demonstrates that thecomplex can cleave specifically at a left-handed site.

Other conformations are not similarly accessible to the chiral cobaltcomplex. Λ-tris(diphenylphenanthroline) complexes of ruthenium(II) andcobalt(III) do not react as assayed spectrophotometrically (34,63) (forruthenium) and by cleavage assays (64) (for cobalt) with B-form helices.The delta isomer in contrast can bind both B- and Z- forms andphotolysis experiments using Δ-Co(DIP)₃ ³⁺ show non-specific cleavage ofthe linear DNA but with some specific band formation. It has also beenfound that (phen)₃ Ru²⁺ complexes do not bind significantly todouble-stranded RNA and hence it is unlikely that Λ-Co(DIP)₃ ³⁺ wouldrecognize an A-form helical conformation Additionally, racemic Co(DIP)₃³⁺ cleavage of single-stranded phage DNA X174 was examined. Here afterphotolysis about 12% cleavage was observed, less than the 15%double-stranded content in the X174 sample, calculated based uponhypochromicity. It is unlikely then that Co(DIP)₃ ³⁺ enantiomers couldrecognize open looped regions of a plasmid. Instead the only DNAconformation for which appreciable binding and cleavage by Λ-Co(DIP)₃ ³⁺has been found is Z-DNA.

Photolysis and digestion of both pLP32 and pBR322 actually yieldsseveral distinct fragments, seen in FIG. 12, and therefore additionalcleavage sites for Λ-Co(DIP)₃ ³⁺, similar structurally to theleft-handed d(CG)₃₂ insert must be present in these plasmids. Based uponnumerous trials using either EcoRI, BamHI, AvaI, or NdeI forlinearization, there appears to be four discrete cleavage sites inpBR322, given in order of intensity 1.45±0.05 kb, 3.3±0.1 kb>4.24±0.02kb>2.25±0.07 kb. The standard deviations are based upon averaging atleast seven experiments. The plasmid pLP32 shows cleavage at these samepositions in addition to cleavage at the insert. FIG. 13A shows atypical EcoRI digest. Fragment pairs are evident and, since EcoRIlinearizes at the origin, the lengths of one fragment of the pair showsthe position in kilobases of the site. The relative intensities,weighted by the fragment molecular weight, reflects either the relativesite affinity for Λ-Co(DIP)₃ ³⁺ or relative cleavage efficiency at asite. Variations in relative site intensities as a function ofirradiation time and also as a function of salt concentration in theincubation mixture were observed. The influence of salt and superhelicaldensity on the relative expression of these sites is currently beingexamined. The weakest site recognized is consistently at 2.3 kb. Table 1summarizes the specific sites in pBR322 found with cleavage byΛ-Co(DIP)₃ ³⁺.

                  TABLE 1                                                         ______________________________________                                        Λ-Co(DIP).sub.3.sup.3+  Cleavage Sites                                            Alternating                                                                   Purine-Pyrimicine                                                             Sequences                                                          ______________________________________                                        1.45 ± 0.05 kb                                                                          1447-1460                                                                     CACG .sub.--GGTGCGCATG                                           2.25 ± 0.07 kb                                                                          2315-2328                                                                     CGCACA .sub.--GATGCGTA                                           3.32 ± 0.11 kb                                                                          3265-3277                                                                     GTATATATG .sub.--AGTA                                            4.24 ± 0.02 kb                                                                          4254-64                                                                       T .sub.--CCGCGCACAT                                              ______________________________________                                    

V. In vitro Screening

Diphenyl tris complexes of this invention were screened for cytotoxicactivity against mouse leukemia cells as previously described. Theresults for two of the complexes are set forth in Table 2.

                  TABLE 2                                                         ______________________________________                                        Cytotoxicity of Cobalt and Ruthenium Complexes                                Compound      Cell Line.sup.1                                                                         ID.sub.50 (μg)/ml)                                 ______________________________________                                        Ru(DIP).sub.3 Cl.sub.2                                                                      L 1210    4.49                                                                P 815     5.42                                                  Co(DIP).sub.3.                                                                              L 1210    0.51                                                  (d-tartrate).sup.3                                                                          P 815     0.52                                                  ______________________________________                                         .sup.1 L 1210 and P 815 are mouse leukemia cell lines, see Burchenal, J.H     et al., CANCER RESEARCH, 42:2598-6000 (July 1982)                        

Discussion

I. Tris(phenanthroline)ruthenium(II)

Results of experiments described above indicate thattris(phenanthroline)ruthenium(II) binds to DNA by intercalation. Theoptical changes on binding to DNA agree with those seen for otherintercalators. Hypochromicity in the metal to ligand charge-transfer(MLCT) band is observed and represents an overall 17% decrease inintensity. Stacking interactions with the base pairs lead to hypochromicshifts in the II→II* transitions of organic intercalating dyes, and itis interesting that the II symmetry of the MLCT preserves thehypochromic effect. Substantial increases in the luminescence of (phen)₃Ru²⁺ also accompany binding to the duplex. The enhancement in emmissionand corresponding increased luminescent lifetimes may simply reflect thedecreased mobility of the complex when sandwiched into the helixEmission lifetimes are comparable to those found for (phen)₃ Ru²⁺ insodium lauryl sulfate micelles.(82). In addition to perturbations in theelectronic structure of the bound reagent, intercalation leads tohydrodynamic changes in the DNA duplex. With increasing concentrations,(phen)₃ Ru²⁺ reversibly unwinds and rewinds superhelical DNA. Althoughnot absolutely definitive,(4) this result provides a very strongindication of intercalation. Surely helical unwinding and lengtheningaccompany the binding of (phen)₃ Ru²⁺. Finally the binding isothermsobtained by equlibrium dialysis yield parameters that are reasonable forthe intercalative mode of association. The complex binds to duplex DNAwith relatively low affinity and, when bound, encompasses a fourbase-pair site. The octahedral coordination around the metal precludeseffective stacking of the complex between base pairs. If the complex isviewed with one of the three phenanthroline ligands inserted into thehelix, then the other two ligands actually protrude above and below theface of this phenanthroline and decrease the effective area of overlap.Hence only partial insertion is possible, which accounts for the lowbinding constant The fact that so small a region of overlap with onlypartial insertion is necessary for a stabilizing interaction with theduplex is interesting to consider with respect to the binding ofaromatic amino acid residues to DNA. The four base-pair site size issimilarly consistent with the structural model for the bound complex,presented herein, where one ligand intercalates and the remaining twoligands span the groove of the helix. A site size of four base pairs isunderstandable since the internuclear distance of 10.4 A between distalhydrogen atoms on the ligands not only exceeds the 10.2 A of a singleinterbase pair site but must result in partial blockage of the nextneighboring base pair both above and below the plane of insertion.

Furthermore the direct comparison between enantiomers of spectroscopicfeatures, binding properties, and structural parameters establishes thatthe Δ enantiomer possesses the greater affinity for a right-handedhelix. Intercalation of tris(phenanthroline)ruthenium(II) into theduplex imposes different steric constraints on Δ and Λ isomers, and itis this difference that determines the enantiomeric selectivity. Perhapsthe strongest evidence in support of intercalation is the observedchiral discrimination. The Δ enantiomer, a right-handed propeller-likestructure, displays a greater affinity than Λ-(phen)₃ Ru²⁺ for theright-handed DNA helix FIG. 14 illustrates the basis for theenantiomeric selectivity With one phenanthroline ligand intercalated,the two nonintercalated ligands of the Δ isomer fit closely along theright-hand helical groove. The nonintercalated ligands of the Λenantiomer, in contrast, are repelled sterically by the phosphatebackbone of the duplex. The disposition of the left-handed enantiomer isopposed to the right-handed helical groove The stereoselectivity seenhere is in the direction proposed originally for (phen)₃ Zn²⁺ andsupports the assignment of the absolute configurations for the zincenantiomers No stereoselectivity is apparent in the association of(phen)₃ Ru²⁺ with Z-DNA. This left-handed helix does not contain agroove of size and depth comparable to that in B-DNA, and thereforecomparable or actually mirror image steric constraints are not expectedInstead the base pairs in the Z-DNA helix are pushed outward toward thesolvent, resulting in at most a very wide and shallow major "groove".Hence Z-DNA provides a poor template for this discrimination.

Although it is Δ-(phen) Ru2+that binds preferentially to B DNA, the Λenantiomer does intercalate into the right-handed helix. The ratio ofaffinities of Δ and Λ isomers for B-DNA is 1.1-1.5, depending upon themethod of analysis Luminescence enhancements and unwinding experimentswith supercoiled DNA suggest the Δ isomer to bind 30-50% more strongly.It is interesting that supercoiling does not alter the selectivity.Optical enrichment assays, which can be extremely sensitive and reflecta direct competition between enantiomers for the helix, yield values of10-30% greater affinity of Δ-(phen)₃ Ru²⁺ for calf thymus DNA. A moreprecise determination of relative affinities is difficult because thebinding constant of either enantiomer for the helix is low. In fact,then, the enantiomer can bind to the right-handed helix, although thephosphate backbone limits access. The addition of bulky sustituents ontothe phenanthroline rings, in severly blocking interactions of theleft-handed enantiomer with the duplex, is necessary to preventcompletely intercalation of the Λ isomer.

These results provide an example of stereospecific interactions withDNA. The stereoselectivity observed is governed by the handedness of theDNA helix. The asymmetric duplex structure serves as a template whichdiscriminates in binding the small molecules on the basis of theirchirality. It is interesting that the change in symmetry of the metalcomplex alone yields a significant difference in its recognition by thehelix. The comparison of spectroscopic and binding characteristics ofisomers of (phen)₃ Ru²⁺ has afforded a detailed description of thestructural basis for the enantiomeric selectivity observed first for(phen)₃ Zn²⁺ (15). The difference in biological activities oftris-(phenanthroline)metal enantiomers is, perhaps, also a function ofthis stereoselectivity.(83) Indeed the interaction of (phen)₃ Ru²⁺ withDNA illustrates how stereospecificity may be incorporated into thedesign of drugs that bind to the duplex and provides a means to designreagents that can distinguish the handedness of the DNA helix.(35,63,64) Certainly these stereospecific interactions underscore theability of small intercalating drugs to recognize differences in nucleicacid structure.

II. Tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II)

The chiral DIP complexes, as shown by the experiments described aboveserve as specific chemical probes for the handedness of the DNA helix insolution. Spectrophotometrics titratiors have shown that, although oneRuDIP enantiomer, assigned as Λ-RuDIP, does not bind at all to the B-DNAhelix, the bulky asymmetric cation can bind to Z-DNA. Monitoring thebinding of this isomer to DNA by any means therefore equivalently assaysthe helical conformation. The intense metal-to-ligand charge-transferband in the ruthenium complexes provides a particularly sensitive handlewith which to examine the binding, either spectrophotometrically orthrough its accompanying luminescence.

Striking enantiometic selectivity is found in the interactions of theRuDIP cations with right-handed B-DNA, and this chiral discrimination isconsistent with an intercalative model. The changes in the visiblespectrum of RuDIP, i.e., the hypochromic shift and luminescenceenhancement observed in the presence of duplex DNA parallel in detailthose seen in spectra of (phen)₃ Ru²⁺ as a function of DNA addition. Ithas been shown that (phen)₃ Ru²⁺ binds to B-DNA by partial intercalationof the phenanthroline ligand between the base pairs (34,35). Given thatgenerally the ruthenium-metal-to-ligand charge-transfer transition showslittle sensitivity to solvent or environment (84,85), the closeresemblance of properties of RuDIP to (phen)₃ Ru²⁺ suggests that thecations bind the DNA in a similar fashion. Intercalation of thediphenylphenanthroline ligand between the helix base pairs requires thatthe phenyl groups rotate into the plane of the phenanthroline ligand.This rotation to a planar structure with minimal steric interactionsbetween nearby hydrogen atoms can be accomplished by legthening thecarbon-carbon bond between the phenyl and phenanthroline moieties.Equivalent structural distortions are seen in biphenyl, which is planarin the stacked solid lattice (86). Also, the extremely bulkytetrapyridyl-porphyrin cations, which require extensive distortation,are thought to bind to the DNA duplex by intercalation (87, 88).Importantly, once rotated into the plane of the phenanthroline, thephenyl groups in RuDIP add substantially to the surface area availablefor overlap with the base pairs as compared with (phen)₃ Ru²⁺, andtherefore greater stability of the bound ruthenium-DNA complex isexpected. In fact, binding of RuDIP to DNA assayed by any method becomesevident at 10% of the concentration of (phen)₃ Ru²⁺, which reflects theincreased affinity of RuDIP over (phen)₃ Ru²⁺ for B-DNA. Perhaps theclearest support for the intercalation model rests in the dramaticenhancement in stereoselectivity observed for RuDIP in comparison with(phen)₃ Ru²⁺. The phenyl groups, while facilitating intercalation ofA-RuDIP into the right-handed helix, completely preclude binding by theΛ enantiomer.

Corey-Pauling-Kolton space-filling models of the RuDIP complexes with aB-DNA helix are shown in FIG. 15. The orientations with respect to thehelix are indicated in the accompanying sketches. The Λ enantiomer, withone diphenylphenanthroline ligand intercalated, can fit very closelyalong the helical groove. The two nonintercalating ligands, with adisposition in line with the right-handed helix, abut the helicalgroove. These close hydrophobic interactions of the nonintercalatedligands are not possible with the mirror-image enantiomer. In contrast,as presented in FIG. 14, if one ligand (not visible) is orientedperpendicular to the helix axis, then the two remaining ligands of the Λenantiomer are disposed contrary to the right-handed groove. Theruthenium model must therefore be shown in front of the DNA helix in thefigure, rather than intercalated, because the interaction of the phenylgroups with the DNA-phosphate backbone at the positions indicated by thearrows completely blocks access. Thus, the stereoselectivity that we seeis determined by the steric constraints imposed by the asymmetry in thehelix, its handedness.

Just as the helix asymmetry can serve as a template to discriminatebetween RuDIP enantiomers, differential binding by the enantiomers maybe used advantageously in determining the chirality of the helix. Table3 indicates a general scheme to probe helical conformations using RuDIPcations. Although Λ-RuDIP does not bind to the right-handed B-DNAduplex, spectrophotometric titrations have shown significant binding toZ-DNA and therefore hypochromism of Λ-RuDIP on addition of a test DNAsample may be used as an indication of the Z-conformation. It wasparticularly interesting to us to find that no stereoselectivity governsbinding to the Z-form helix. The bulky cation likely avoids the verynarrow helical crevice in the Z-DNA structure, and intercalative bindingto the more shallow hydrophobic surface in Z-DNA, the equivalent of themaJor groove in the B-form, would not be expected to yield any chiraldiscrimination. Z-DNA does not mirror B-DNA in solution. Instead wepredict that a left-handed but more B-like conformation (18, 89, 90)would yield a mirror-image selectivity.

Table 3. Scheme for probing DNA conformation with RuDIP enantiomers

                  TABLE 3                                                         ______________________________________                                        Scheme for probing DNA conformation with RuDIP                                enantiomers                                                                   Reactivity                                                                    With the With tje                                                             Δ isomer                                                                         Λ isomer                                                                          DNA duplex conformation                                   ______________________________________                                        +        -          Right-handed B-like                                       +        +          Left-handed Z-like or lacking a                                               groove                                                    -        -          Unstacked or with base pairs                                                  inaccesible                                               -        +          Left-handed B-like                                        ______________________________________                                    

The chiral tris(diphenylphenanthroline) metal complexes may therefore beused in solution to examine DNA helical conformations: those ofnaturally occurring sequences, in the presence of drugs, and inprotein-bound complexes. Furthermore, the reagents represent a new routefor conformation-specific drug design.

III. Cobalt(III) Complexes

The DNA cleavage experiments described above are important in severalrespects The photoactivated DNA cleavage reaction with Co(phen)₃ ³⁺illustrates with a simple inorganic complex the notion of DNA strandscission mediated by a locally generated redox reaction. Reduction ofCo(III) with perhaps concomitant hydroxide oxidation may be responsiblefor cleavage. With regard to applications, this photoactivated reactionshould make possible "footprinting" as a function of time. Mostimportantly, the differential cleavage of ColEl DNA by enantiomers ofCo(DIP)₃ ³⁺ represents a clear example of a conformation-specific DNAcleaving molecule. This molecule will be useful in determining regionsof Z-DNA conformation within long segments of native DNA. Moreover thehigh level of recognition of DNA conformation by these chiral inorganiccomplexes suggests the powerful application of stereospecificity in DNAdrug design.

IV. Cleavage Site Mapping

Features unique to the plasmid recognition sites determined above withΛ-Co(DIP)₃ ³⁺ were examined. Λ-Co(DIP)₃ ³⁺ is not a sequence-specificreagent and there is no sequence homology evident at these positions inpBR322. Instead Λ-Co(DIP)₃ ³⁺ is a conformation-specific cleavage agentand it is the common left-handed conformation at these locations that islikely to be recognized by the cobalt complex. Alternatingpurine-pyrimidine sequences have been shown to adopt the Z-DNAconformation most readily, because alternative residues in Z-DNA havebases in the syn conformation. Inspection of the pBR322 sequencerevealed that the Λ-Co(DIP)₃ ³⁺ recognition sites included the longestruns of alternating purines and pyrimidines allowing for one base out ofalternation. Table 1 shows also the alternating sequences that appearwithin one standard deviation of each measured recognition site. Atpositions 1447, 2315, 3265, and 4254 begin respectively 14, 14, 13, and11 base pair regions with alternating purine and pyrimidine residueshaving one mistake. These regions correspond essentially to one helicalturn in a Z-DNA conformation and are the longest of such conformationhomology within the plasmid. Sequences not recognized by Λ-Co(DIP)₃ ³⁺were then considered. There are several other sequences, beginning at1171, 1533, and 1709, that also constitute 11 base pair segments withone mistake that are not significantly cleaved by Λ-Co(DIP)₃ ³⁺, and thelongest sequence of alternation in the plasmid with no mistakes, 10 bpbeginning at position 2785, is also not cleaved. At this stage it is notknown whether the flanking sequences at these sites are affecting Z-DNAformation or Λ-Co(DIP)₃ ³⁺ recognition. The sequences within therecognition sites detected do have a range of GC contents. It isinteresting that the Z-DNA conformation in pBR322 has been detected atthe 1447 site in equally low salt buffers by a completely differentroute, crosslinking studies (80) with anti-Z-DNA antibodies, which lendsconfirmation to the Z-form assignment. Thus it is proposed that thesefour sites of alternating purinepyrimidine residues adopt theZ-conformation under physiological conditions in native supercoiledpBR322 and are specifically recognized and cleaved by Λ-Co(DIP)₃ ³⁺.

It is interesting, finally, to ask whether these Z-DNA segments sharesome common biological function in this plasmid. pBR322, assembled fromthree naturally occurring plasmids, contains three genetically distinctcoding regions, the tetracycline resistance genes, the β-lactamase geneconferring ampicillin resistance, and the origin of replication. FIG.13B shows the map of these genes in pBR322. It is curious to notice thecorrespondence in position between the ends of these discrete codingelements and the Z-DNA recognition sites. A single polypeptide in pBR322appears to be necessary for tetracycline resistance (91). The 3'-end ofthe region encoding this peptide is thought to be near the AvaI site at1425 bp; sequences upstream from the tetracycline resistance promotor(which begin at 45 bp) were lost in construction from pSC101. Theβ-lactamase gene is defined upstream by the start site at 4201 with the-35 consensus region for the promoter ending at 4236, 18 bp away fromthe Z-form cleavage site (92). The 3'-end of the region encodingβ-lactamase is found at position 3295, which is 22 bp upstream of theZ-form alternating purine-pyrimidine site. Lastly the essential regionconstituting the origin of replication in pBR322 extends from theRNA/DNA junction at 2536 to position 2360, 32 base pairs from the weakZ-form site detected with Λ-Co(DIP)₃ ³⁺ (65,93). Thus there appears tobe a remarkable correspondence between Z-DNA sites recognized by thecobalt complex and the ends of genetic coding elements. It is temptingto suggest that the Z-DNA conformation might provide a generalstructural signal or punction mark which demarcates the ends of thesegenes. Consistent with this idea, the Z-conformation has been shown to.provide a poor template for transcriptional activity with E coli RNApolymerase (94). This notion is consistent also with the location ofalternating purine-pyrimidine tracts in SV40 DNA enhancer sequenceswhich bind anti-Z-DNA antibodies (30), to the d(GT)n tracts at the endsof yeast chromosomes (95), and to the alternating purine-pyrimidine longterminal repeats in mouse mammary tumor virus (96). Moreover recentexperiments with mung bean nuclease in the malaria parasite Plasmodiumhave demonstrated that a particular conformation rather than a sequenceappears to be shared by gene termination sites (97). These correlationsof location with conformation are intriguing and support the notion thatDNA polymorphism may be involved in gene expression, that specificsequences may adopt specific local conformation which containinformation, and that chromosomal regulation may involve DNAconformation-specific signals. In sum, the results presented hereinindicate that several discrete Z-DNA sites exist under nativephysiological conditions in pBR322 The positions of these sites mark theends of genetically distinct coding elements in the plasmid. A cobaltcomplex of this invention, e.g. Λ-Co(DIP)₃ ³⁺ thus provides aphotoactivated site-specific cleaving agent that is useful to map thesesites and should be helpful in establishing a relationship between thelocations of Z-DNA segments and its biological function.

V. Anti-tumor Activitv

Complexes of this invention showed high potency against leukemia cellsin the previously described screen. These complexes should be useful asanti-tumor agents, and should be active in vivo as well, e.g. in acomposition containing a pharmaceutically acceptable carrier.

The results set forth in Table 2, above also indicate a synergisticeffect when use of a cobalt complex is combined with ultravioletradiation. A decrease in the amount of cobalt required for cell death ofgreater than about 10-fold, and in some cases up to 50-fold has therebybeen observed.

Isolation of chromatin slowed extensive DNA cleavage. Hence it appearsthat the complexes can pass into the cell, remain intact therein andinteract with DNA as a cellular target.

Since these complexes contain large, planar ligands, selectiveintercalation is optimized. Individual enantiomers and mixed-ligandcomplexes should also be useful in this and other embodiments of theinvention. The Λ Z-specific enantiomers should be especially useful inanti-tumor compositions and uses. cl References (I)

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What is claimed is:
 1. A coordination complex having the formula (R)3--- CO(III), wherein R is 1,10-phenanthroline or a substitutedderivative thereof selected from the group consisting of4,7-diamino-1,10-phenanthroline; 3,8-diamono-1,10-phenanthroline;4,7-diethylenediamine-1,10-phenanthroline;3,8-diethylenediamine-1,10-phenanthroline;4,7-dihydroxyl-1,10-phenanthroline; 3,8-dihydroxy-1,10-phenanthroline;4,7-dinitro-1,10-phenanthroline; 3,8-dinitro-1,10-phenanthroline;4,7-diphenyl-1,10-phenanthroline; 3,8-diphenyl-1,10-phenanthroline;4,7-dispermine-1,10-phenanthroline; and3,8-dispermine1,10-phenanthroline; and R is bound to Co by acoordination bond or salt thereof.
 2. The optically resolved Δ isomer ofa complex of claim
 1. 3. The optically resolved Λ isomer of a complex ofclaim 1.